DESCRIPTION: Antibacterial resistance is a serious and growing public health threat. In the United States, each year at least 2 million people become infected with bacteria that are resistant to antibiotics and at least 23,000 people die as a direct result of these infections. Pseudomonas aeruginosa, an antibiotic resistant bacterium commonly found in soil and water, has been recognized as one of the most commonly-isolated nosocomial pathogen accounting for over 10 percent of all hospital-acquired infections. Understanding bacterial extracellular interaction of P. aeruginosa and its response to drug exposure is thus highly desired and important. The long-term goals of our research are (1) to develop an effective optical tweezers technique that can be used to quantify the bacterial- bacterial cell interaction and the stimulus-response relation; and (2) to build up a bacterial chemotaxis database that will be useful for understanding antibacterial resistance and for drug development. The objective of the current application is to design and set up an optical tweezers for manipulating especially rod-shaped bacteria such as a P. aeruginosa cell, and for investigating its aggregation with neighboring cells and interaction with different stimuli (drugs). We hypothesize that a rod-shaped bacterium can be stably trapped in three dimensions yet examined in an observing plane by a tug-of-war optical tweezers (which can apply much stronger pulling forces than that in conventional tweezers), and its clustering with neighboring cells and response to a stimulus can be quantitatively measured. Guided by strong preliminary data, these hypotheses will be tested through the following Specific Aims: (1) Design and set up an optical tug-of-war tweezers system that allows for stable in- plane trapping and stretching of rod-shaped bacteria. Such a system with dark-field microscopy enables high- contrast imaging and direct monitoring the bacterial motility without the need of fluorescent labeling. (2) Employ the tug-of-war tweezers to study bacterial extracellular interaction with two specific examples. The first is adhesive Sinorhizobium meliloti bacterial cells grown under different growth media (say PYE vs. TY), and the second is P. aeruginosa bacterial clusters prepared with or without Pel polysaccharide. This novel tweezers with tunable pulling forces will be used to characterize stable and unstable cell aggregations in these two cases, thus identifying the role of a particular medium in bacterial-bacterial cell interaction and cellular aggregation. (3) Employ the tug-of-war tweezers to study bacteria-drug interaction. By varying the strength (doses) and/or the frequency of exposure to a drug (e.g., Colistin), the motion of a P. aeruginosa cell in the optical trap due to bacteria-drug interaction can be quantified through direct chemotactic force measurement. Successful completion of these studies will help us understand bacterial extracellular interaction in biofilm formation and bacterial resistance to drug exposure. Thus, it will have significant clinical impact